Antibiotic cirolerosus and process for producing the same



Oct. 3, 1967 c. E. MEYER ETAL 3,345,262

ANTIBIOTIC CIROLEROSUS AND PROCESS FOR PRODUCING THE SAME Filed March16. 1964 2 Sheets-Sheet l ooom 00.9 Oo oo oom 003 002 002 ooh 009 O 3 (Duolsslwsuvan m:m omm om u mo zzmhommw amm mmz mmzmE B. K. BHUYAN c; E.MEYER INVENTORS ATTORNEYS 1967 c. E. MEYER ETAL 3,345,262

ANTIBIOTIC CIROLEROSUS AND PROCESS FOR PRODUCING THE SAME Filed March16, 1964 2 Sheets-Sheet 2 0 E I I l O -m x w I o I o q- I (O O' l U) INO I x o L!) i i J l E o 0 Lu 2 z O o J I I In o rr- Lu Lu E t: g 1

I i l IN V EN TORS United States Patent 5,345,262 IOTIC CROLERCSUS ANDPROCESS FOR PRODUCING THE SAME Curtis E. Meyer, Charleston Township,Kalamazoo County, and Bijoy K. Bhuyan, Kalamazoo, Mich., assignors toThe Upjohn Company, Kalamazoo, Mich, a corporation of Delaware FiledMar. 16, 1964, Ser. No. 352,257 Claims. (Cl. 167-65) ABSTRACT OF THEDISCLOSURE New antibiotic cirolerosus and a microbiological process forthe production thereof. Cirolerosus can be used to inhibit the growth ofvarious bacteria, for example, Bacillus subiilis, Staphylococcus albus,and Streptococcus lactis.

This invention relates to a novel composition of matter and to processfor the production thereof. More particularly, this invention relates toa new compound, cirolerosus (U-l2241), and to a process for theproduction thereof.

Cirolerosus is a biosynthetic product obtained by culturing acirolerosus-producing actinomycete in an aqueous nutrient medium. It isa basic substance which has the property of adversely affecting thegrowth of certain organisms, particularly bacteria, for example,Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Sarcinalutea, Klebsiella pneumoniae, Lactobacillus casei, Escherichz'a coli,and Mycobacterium avium, and can be used alone or in combination withother antibacterial agents to prevent the growth of, or reduce thenumber of, such organisms present in various environments. For example,it is useful for controlling the infection of silkworms caused "bypathogenic cultures of B. subtilis; it is also useful as an oilpreservative, for example, as a bacteriostatic agent for inhibiting thegrowth of certain microorganisms that cause spoilage in cutting oils.Cirolerosus can be used either as the free base or as an acid additionsalt, alone or in combination with other antibacterial agents, toprevent the growth of or to reduce the number of microorganisms presentin various environments, for example, in plants and in animals such asmammals, birds, fish, and reptiles, where the infecting microorganism issusceptible to the antibiotic. Also, it is useful in wash solutions forsanitation purposes, as in the washing of hands and the cleaning 'ofequipment, floors, or furnishings of contaminated rooms or laboratories;it is also useful as an industrial preservative, for'example, as abacteriostatic rinse for laundered clothes and for impregnating paperand fabrics; and it is useful for suppressing the growth of sensitiveorganisms in plate assays, and other biological media. It'can also beused as a feed supplement to promote the growth of animals, for example,mammals, birds, fish, and reptiles.

THE MICROORGANISM The actinomycete'used according to this invention forthe production of cirolerosus has been designated as Streptomyces bellusvar. cirolerosus. A culture of the living organism can be obtained fromthe permanent collection of the Northern Utilization and ResearchDivision, Agricultural Research Service, US. Department of Agriculture,Peoria, 111., U.S.A. Its accession number in this repository is NRRL3107.

Streptomyces bellus var. cirolerosus, NRRL 3107, is very closely relatedto S. bellus. Both organisms are characterized by predominately blueaerial growth and a red reverse. They are melanin positive, and warty tospiny spores are produced in spiral sporophores. Both Peptone-IronBennetts CIR OLER OS US AND STREP TOZMYC'ES BELL US 0 N E KTACH ROME 1Cultures S. bellus var. cirolerosus Agar Medium S. bellus SurfaceReverse Surface Reverse Blue do Red-tan. Orange Red. Brown. Do. Red-tan.Yellowtan.

Blue l Red-tan Blue-pink Red Yellowtan.

Czapeks Sucrose.

Maltose Tryptone Peptone Iron 0.1% Tyrosine. Casein Starch 1 Dietz, A.,Ektachrome Transparencies as Aids in Actinomycete Classification, Annalsof the N .Y. Academy of Science 60: 152-154, 1954.

UTILIZATION OF CARBON COMPOUNDS BY STREPTOMYCES BELL US VA R. CIR OLEROS US 1 Allgllgltis'lllc/lr'i EPTOMY C'ES BELLUS IN SYNTHETIC S. bellusvar. cirolerosus 1S. bellus Control D-xyl0se L-arabinose. Rharnnose.

D-fructose D-galactose GlyceroL Dulcitol 1- 1- 1- 1- 1-u-n-u-w- 1- s e sz w rps s r esw A A l I I I l l l I v v Na Succinate Pridham, T. o. andGottlieb, 13., Assimilation of Car bon Compounds in Synethic Medium, .T.Bact. 56. 107-114 1948 I Positive Utilization.

' Negative Utilization.

() Slight GrowthNo Utilization.

(+) Positive Utilization0nly Slight Growth.

CULTURAL CHARACTERISTICS OF STREPTOMYC'ES VAR. CIR OLER OSUS ANDSTREPTOMYC'ES E U Medium S. bellus var. ciralerosus S. bellus Trace grayaerial growth.

Brown vegetative growth. Melanin positive.

White aerial growth with trace of blue.

Agar.

Calcium Malate Agar.

Medium S. beZZus var. cirolerosus S. bellus Glucose Asparagine Agar.

X anthlne Agar Pink aerial growth.

Salmon reverse. Very slight trace salmon pigment.

Gray pink aerial growth. Pink tan reverse. Pink tan Peach-white aerialgrowth. Orange Reverse. Pale peach pigment.

No aerial growth. Peach vegetative growth. Peach pigment. Xanpigment.Xanthine thine solnbilized. solubilized. Casein Starch Blue gray aerialgrowth. Blue aerial growth.

Agar. Yellow reverse. Trace Yellow reverse. Trace yellow pigment. yellowpigment. Starch hydrolyzed. Starch hydrolyzed. Tyroxine Agar. Gray pinkaerial Lavender pink aerial growth. Brown regrowth. Pink-tanverse. Brownpigbrown reverse. Pinkinent. Tyrosine solutan-brown pigment. bilized.Tyrosine solubilized. Tomato Paste- Gray-pink aerial Trace pink aerialOatmeal Agar. growth. Rose regrowth. Peach reverse. No pigment. verse.Trace peach pigment. Bennett's Again-.. Lavender-gray aerial Blue aerialgrowth with growth. Red tan retrace of pink. Roseverse. Tan pigment. tanreverse. Pale tan pigment. Czapeks Sucrose Lavender-rose aerial Pinkaerial growth. Agar. growth. Purple re- Rose reverse. No

verse. Trace purple pigment. pigment. Maltose Tryp- Pink aerial growth.Blue-pink aerial growth.

tone Agar. Red-tan reverse. Rose-tan reverse.

Trace tan pigment. Pale tan pigment. Plain Gelatin Tan brown pigment inBrown pigment at surof medium. Liquefied in pigment face. Tan pigment inof medium.

area. Trace liquefaction. Nutrient Gelatin Tau brown pigment in Tracetan pigment. No

M of medium. Liqliquefaction. uefied in pigment area. Synthetic NitrateSurface pellicle. Pink Very slight trace Broth. tan pigment. Reducbottomgrowth. No

tion. reduction. Nutrient Nitrate Surface pellicle. Tan Trace surfacegrowth.

Broth. p gnent. No redue Flocculent at base.

tion. Yellow tan pigment. v No reduction. Litmus Milk Surface ring andpel- Surface ring. No pepliele. Litmus reduced slightly. Nopcptonization. pH 6.2.

tonization. pH 6.1.

COLOR COMPARISON OF S. BELLUS VAR. CIROLEROSUS AND S. BELLUS (COLORSACCORDING TO THE ISCC- NBS METHOD OF DESIGNATING COLORS AND A DIC-TIONARY OF COLOR NAMES, NBS CIRCULAR 553, 1955) The new compound of theinvention is produced when the elaborating organism is grown in anaqueous nutrient medium under submerged aerobic conditions. It is to beunderstood also that for the preparation of limited amounts surfacecultures in bottles can be employed. The organism is grown in a nutrientmedium containing a carbon source, for example, an assimilablecarbohydr-ate, and a nitrogen source, for example, an assimilablenitrogen compound or protcinaceous material. Preferred carbon sourcesinclude glucose, brown sugar, sucrose, glycerol, starch, corn starch,galactose, dextrin, molasses, and the like. Preferred nitrogen sourcesinclude cornstecp liquor, yeast, autolyzed Brcwcrs yeast with milksolids, soybean meal, cotton seed meal, corn meal, milk solids,pancreatic digest of casein, distillcrs solubles, animal pcptoneliquors, meat and bone scraps, and the like. A combination of thesecarbon and nitrogen sources can be used advantageously. Trace metals,for example, zinc, magnesium, manganese, cobalt, iron, and the like neednot be added to the fermentation since tap water and unpurifiedingredients are used as media components.

Production of the compound of the invention can be effected at anytemperature conducive to saisfactory growth of the microorganism, forexample, between about 18 and 40 C. and preferably between about 26 and30 C. Ordinarily, optimum production of the compound is obtained inabout 2 to 10 days. The medium normally stays fairly close to neutral,or on the acid side during the fermentation. The final pH is dependent,in part, on the buffers present, if any, and in part on the initial pHof the culture medium which is advantageously adjusted to about pH 6-8prior to sterilization.

When growth is carried out in large vessels and tanks, it is preferableto use the vegetative form, rather than the spore form, of themicroorganism for inoculation to avoid a pronounced lag in theproduction of the new compound and the attendant incfficicnt utilizationof equipment. Accordingly, it is desirable to produce a cgetativcinoculum in a nutrient broth culture by inoculating the broth culturewith an aliquots from a soil or slant culture. When a young, active,vegetative inoculum has thus been secured, it is transferred asepticallyto large vessels or tanks. The medium in which the vegetative inoculumis produced can be the same as, or different from, that utilized for theproduction of the new compound, as long as it is such that a good growthof the microorganism is obtained.

The new compound of the invention is a basic compound having theempirical formula C45 52H52 76N2O18- It is a red solid and can serve asan indicator in that it is red in acid solution and blue in alkalinesolution. It is soluble in lower alkanols, e.g., methanol, ethanol, andthe like; lowcr-alkyl esters of lower alkanoic acids, c.g., ethylacetate, and the like; halogenated lowcr-alkanes, e.g., methylenechloride, chloroform, ethylene dichloride, and the like; inorganic andorganic acids, benzene and glycols. It is slightly soluble in other andrelatively insoluble in paraffin hydrocarbons.

A variety of procedures can be employed in the isolation andpurification of cirolerosus, for example, solvent extraction,liquid-liquid distribution in a Craig apparatus, and the use ofadsorbents.

Solvent extraction procedures are preferred for commercial productioninasmuch as they are less time consuming and less expensive, and higherrecovery yields are obtained thereby.

In a preferred recovery process, the mycclium and undissolved solids arefirst separated from the fermentation beer by conventional means such asfiltration with the use of a filter aid (or by ccntrifugation). Thefiltered beer (or centrifuged beer) is adjusted to a pH of about 8.5 andextracted with methylene chloride. The methylene chloride extract isconcentrated and the concentrate is extracted with aqueous acid at a pHof about 3.5. The acid extract is adjusted to a pH of about 5.6 withalkali and extracted with methylene chloride. Concentration of themethylene chloride extract and addition of Skellysolve B (isomerichexanes) to the concentrate precipitatcs cirolerosus as a red solidwhich is further purified by counter-current distribution in a systemcomposed of equal parts of toluene, methylene chloride and propyleneglycol. If desired, the solvent phase can be acidified, and the newcompound recovered in a protonated form.

The new compound of the invention can also be recovered from thefiltered beer by adsorption on cation exchange resins. Both thecarboxylic and sulfonic acid types can be used. Suitable carboxylic acidresins include the polyacrylic acid resins obtained by thecopolymcrization of acrylic acid and divinylbenzcnc by the proceduregiven on page 87 of Kum'n, Ion Exchange Resins, 2nd ed. (1958), JohnWiley and Sons, Inc. Carboxylic acid cation exchange resins of this typeare marketed under the trade names Amberlite IRC-SO and Zeokarb 226.Suitable sulfonic acid resins include nuclear sulfonated polystyreneresins cross-linked with divinylbenzene which are obtained by theprocedure given on page 84 of Kunin, supra. Sulfonated cation exchangeresins of this type are marketed under the tradenames Dowex-50,Amberlite IR-120, Nalcite HCR, Chempro C-20, Permutit Q, and Zeokarb225.

The antibiotic is eluted from the'resin with an acid, advantageously ata pH lower than the pKa of the cation exchange resin used. Satisfactoryresults are obtained with a pH of about 1 to 6. The eluate is adjustedto about pH 7.5 to 8.5 with a base, e.g., sodium hydroxide, or astrongly basic anion exchange resin, and the antibiotic. is extractedwith a Water-immiscible solvent according to the process describedabove. [Suitable anion exchange resins for this purpose are obtained bychloromethylating by the procedure given on pages 88 and 97 of Kunin,supra, polystyrene cross-linked, if desired, with divinylbenzeneprepared by the procedure given on page 84 of Kunin, supra, andquaternizing with trimethylamine or dimethylethanolamine by theprocedure given on page 97 of Kunin, supra. Anion exchange resins ofthis type are marketed under the tradenames Dowex 2, Dowex 20, AmberliteIRA-400, Duolite A-102 and Permutit 8-1.]

The novel compound of the invention can also be recovered from harvestbeers and other aqueous solutions by adsorption on a surface activeadsorbent, for example, Florisil (a synthetic silicate of the typedescribed in US. Patent 2,393,625 and sold by the Floridin Company),decolorizing carbon, or decolo-rizing resins, and eluting the adsorbedmaterial with a solvent. Any of the solvents mentioned above can beused. A suitable decolorizing resin is Permutit DR (US. Patent2,702,263).

The new compound of the invention can be purified by successivetransfers from protonated to non-protonated forms and vice versa,especially with other types of treatments intervening as for example,solvent extractions and washings, chromatography, and fractionalliquidliquid extraction. In this manner salts of cirolerosus can beemployed to isolate or upgrade the antibiotic. For example, theantibiotic can be converted to an insoluble salt, such as the picrate,which can be subjected to purification procedures and then used toregenerate the antibiotic free base by treatment with alkali. Or theantibiotic can be converted to a water-soluble salt, such as thehydrochloride or sulfate, and the aqueous solution of the salt extractedwith various water-immiscible solvents before regenerating theantibiotic free base by treatment with alkali of the thus-extracted acidsolution.

Salts of cirolerosus can be used for the same biological purposes as thefree base or they can be employed to upgrade the antibiotic aspreviously described.

Specific acid salts can betmade by neutralizing the free base with theappropriate acid to below about pH 7.5, and advantageously to about pH 2to pH 6. Suitable acids, for this purpose include hydrochloric,sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic,furmaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric,glycolic, phthalic, tartaric, lauric, stearic, salicyclic, 3-phenylsalicyclic, 5 -'phenylsalicyclic, 3 methylglutaric,orthosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic,1-2-cyclohexanedicarboxylic, 4-cyclohexenecarboxylic,octadecenylsuccinic, octenylsu'ccinic, methanesulfonic, benzenesulfonic,helitanthic, Reineckes, dimethyldithiocarbamic, sorbic,monochloroacetic, undecylenic, 4' hydroxyazobenzene 4 sulfonic,octadecylsulfuric, picric, benzoic, cinnamic, and like acids.

The new compound of the invention, cirolerosus, has a broad spectrum ofantibacterial activity. On the agar disc plate assay using a 13 mm.disc, cirolerosus is active against the following organisms:Streptococcus lactis, Staphylococcus albus, Bacillus cereus, Sarcimzlutea, Lactobacillus casei, and Bacillus subtilis. It also inhibits thegrowth of KB human epidermoid carcinoma cells in tissue culture. Theprocedure of Smith et al. (Smith, C. G.; Lummis, W. L, and Grady, J. E.:An improved tissue culture assay. I. Methodology and cytotoxicity ofantitumor agents, Cancer Research, 19:8432846, 1959) was used todetermine the cytotoxicity of cirolerosus to KB human epidermoidcarcinoma cells in tissue culture. KB cell protein synthesis wasinhibited 50% (IB at a concentration of 0.004 mcg./ml.

The new compound of the'invention, cirolerosus, is active againstBacillus subtilis and can be used to minimize or prevent odor caused bythis organism in fish and fish crates. The new compound can be used as adisinfectant on various dental and medical equipment contaminated withStaphylococcus albus; it can also be used as a disinfectant on washedand stacked food utensils con taminated with Staphylococcus albus. Thenew compound of the invention is active against Streptococcus lactis,which causes the souring of milk, and can be used to prevent or delaysouring of dairy products, for example, milk, and cheese. The newcompound can also be used to inhibit gram-positive sporeform spreaderson agar plates when isolating molds, yeasts, and streptomycetes.

The new compound of the invention, cirolerosus, gives a red color inacid solution and a blue color in alkaline solution. Accordingly,cirolerosus can be used as a pH indicator.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 .Cir0lerosus '(A) Fermentation.A solid stock of Streptomycesbellus var. cirolerosus, NRRL 3107, was used to inoculate 500 ml.Erlenmeyer flasks containing ml. of sterile pre-seed medium consistingof the following ingredients:

Glucose monohydrate gm 25 Pharmamedia 1 gm 25 Tap water q.s liter 1Pharmamedia is an industrial grade of cottonseed flour produced byTraders Oil Mill Company, Fort Worth, Tex.

The pre-seed medium pre-sterilization pH was 7.2. The pro-seed inoculumwas grown for 2 days at 28 C. on a Gump rotary shaker operating at 260r.p.m.

The pre-seed inoculum (600 ml.) was used to inoculate a 400 liter seedtank containing 250 liters of the following sterile seed medium:

Gm./l. Glucose monohydrate 10 Corn-steep liquor 10 Pharmamedia 2 WilsonsPeptone Liquor No. 159 l0 Lard oil V 2 Tap water Balance Gm./l. Glucosemonohydrate 2O Starch 20 Suffolk peanut meal 30 Lard oil 2 Tap waterBalance An industrial grade of peanut meal produced by Suffolk Qll Mill,Inc., Suffolk, Va. Y

The pH was adjusted to 6.8 with sodium hydroxide before sterilization.The culture was grown for 4 days at a temperature of 28 C. with aerationat the rate of 40 7 standard cubic feet per minute, and stirring at therate of 166 r.p.m. Lard oil was added during the fermentation to controlfoaming.

(B) Extractin.-Whole beer from a fermentation as described above,assaying approximately '20 biounits of cirolerosus/ml. with about 3.8%solids, is adjusted to pH 2.0 with sulfuric acid and filtered using 2.5%diatomaceous earth as filter aid. The filtered beer is adjusted to pH8.5 with 50% sodium hydroxide and extracted 2 times with /3 vol.portions of methylene chloride. The methylene chloride extracts arecombined and concentrated to /3 vol. under reduced pressure. Themethylene chloride concentrate is mixed with /3 vol. of Water, and thismixture is then adjusted to a pH of about 3.5 with sulfuric acid andextracted 2 times with vol. portions of Water while maintaining the pHof the stirred mixture at about 3.5. The aqueous extracts are combined,adjusted to pH 5.6 with 50% sodium hydroxide, and extracted 2 times with/3 vol. portions of methylene chloride. The methylene chloride extractsare combined and concentrated to A vol. Skellysolve B (isomeric hexanes)is added to the concentrate until a complete precipitation ofcirolerosus is accomplished. The red precipitate is then filtered anddried; yield of cirolerosus (ca. 95% pure) is 390 grams assaying 200biounits/ mg. on a Bacillus subtilis assay. (The assay is an agar plateassay using Trypticase Soy Broth medium with 30 meg/ml. of Mn'CITrypticase is a pancreatic digest of casein. The assay plates are seededwith 0.05 ml. of B. subtilis spore suspension per 100 ml. agar andincubated at 30 C. overnight.) A biounit is that amount of antibioticwhen dissolved in 0.8 ml. of the test solution and applied to a 12.7 mm.disc gives a 20 mm. zone of inhibition under standard mirobiologicalconditions. (One microgram of substantially pure cirolerosus assays0.211 biounit.)

(C) Purificari0n.-Cirolerosus, 5.5 grams obtained as above, wasdissolved in 500 ml. of a solvent system composed of equal parts oftoluene, methylene chloride, and propylene glycol. This solution wasplaced in tubes 0-24 (1 0 ml. of each phase per tube) of a countercurrent distribution apparatus. After 500 transfers, aliquots of theupper and lower phases of every tenth tube were dilutedappr-opriatelywith methylene chloride and analyzed colorimetrically. The contents oftubes 420 through 470 were pooled, the propylene glycol Was removed bywashing with water and the toluene-methylene chloride solution wasconcentrated under reduced pressure. The concentrate was poured intoSkellysolve B and the red precipitate of cirolerosus was collected;yield 3.3 gm.

Example 2.Cir0lerosus hydrochloride One gram of cirolerosus, prepared asin Example 1, was dissolved in 80 ml. of acetone. The dark red solutionwas filtered and 0.16 ml. of concentrated hydro chloric acid was added.The sharp color change from red to light, bright red occurred at aboutpH 5 and after about 0.14 ml. of hydrochloric acid had been added, afterwhich the pH dropped abruptly to 2. The precipitate of cirolerosushydrochloride was filtered and dried; yield 865 mg.

Example 3.Ciroler0sus sulfate To a solution of 550 mg. of cirolerosus,prepared as in Example 1, in 50 ml. of absolute ethanol Was added 3drops of 6 N sulfuric acid. The flocculent precipitate which formed wascentrifuged from suspension and dissolved in 50 ml. of absolute alcoholwith heating. Upon cooling, an amorphous precipitate formed. Theaddition of 75 ml. of ethyl ether precipitated an additional amount ofcirolerosus sulfate which was filtered off and dried; yield 109 mg.

The addition of 2 more drops of 6 N sulfuric acid to the originalsupernatant followed by ethyl ether gave another 138 mg. of cirolerosussulfate.

Example 4..Cir0ler0sus picrate To 400 mg. of cirolerosus, prepared as inExample 1, in 100 ml. of absolute ethanol was added an ethanolicsolution of 100 mg. of picric acid. A dark red precipitate ofcirolerosus picrate which formed immediately was filtered and dried.

Example 5.-Cir0ler0sus 4hydr0xyaz0benzene-4- sulfonate One gram ofcirolerosus, prepared as in Example 1, was dissolved in 100 ml. of waterwith suflicient hydrochloric acid to give a pH of 5.0. A solution of 1.2gm. of 4-hydroxyazobenzene-4-sulfonic acid in 100 ml. of water wasadded, and cirolerosus 4-hydroxyazobenzene- 4-sulfonate separated as afiocculent precipitate. The salt was separated by centrifugation,dissolved in about 200 ml. of ethyl alcohol and reprecipitated by theaddition of 350 ml. of ethyl ether. The cirolerosus4'-hydroxyazobenzene-4-sulfonate was collected by filtration and dried;yield 950 mg.

CHEMICAL AND PHYSICAL PROPERTIES OF CIROLEROSUS Emprical formula: c 5 5H 2 7 N2O13 Elemental analysis:

Acetyl=9.79%

Specific optical rotation: Too highly colored.

Solubility: Soluble in methanol, ethanol, ethyl acetate, halogenatedhydrocarbons, acids, benzene, glycols. Slightly soluble in ether.Relatively insoluble in paraffin hydrocarbons.

Ultraviolet spectrum: The ultraviolet absorption maxima of cirolerosusas reproduced in FIGURE II of the drawing are:

In .01 N acid (alcoholic HCl)-- 236 Ill 2., a=40.33 253 111 3:23.01(sh.) 293 mg, a=8.08 465 61,1, 3:10.11 (sh.) 495 III/1, a=12.15 515 mg,a=12.63 (sh.) 525 m a=12.l5 (sh.) 549 11114., a=5.97 (sh.) 564 III/2.,a=5.23

.01 N base (alcoholic NaOH)- 242 m a=42.9l 292 III/17., a=7.74 580 m,u.,21:14.32 610 111 a=1358 (sh.)

Infrared spectrum: The infrared absorption spectrum of cirolerosussuspended in mineral oil mull is reproduced in FIGURE I of the drawings.Cirolerosus shows peaks at the following wave lengths expressed inreciprocal centimeters:

1377 (S) (oil) 835 (M) 9 1367 (S) 818 (M) 1299 (S) 797 (M) 1251 (S) 771(M) 1241 (S) 746 (M) 1224 (S) 723 (M) 1197 (S) 712 (M) 1170 (S) 703 (M)1115 (S) 663 (W) Band intensities are indicated as S, M, and W,respectively, and are approximated in terms of the backgrounds in thevicinity of the bands. An S band is of the same order of intensity asthe strongest in the spectrum; M bands are between A and as intense athe strongest band, and W bands are less than /3 as intense as thestrongest hand. These estimates are made on the basis of a percenttransmission scale.

Molecular weight: Molecular weight is 974 :30 (from eq. wt.). Titration:

In waterpKa pKa 7.68 In dimethyl-iormam-ide pKa 7.2 PKHZ 8.2

We claim:

.01 N acid (alcoholic HCl)- 236 m n, a=40.33 253 m a=23.01 (sh.) 293 Ill1., a=8.08 465 m n, a=10.11 (sh.) 495 mu, a=12.15 515 m 21:12.63 (sh.)525 m a=12.15 (sh.) 549 m a=5.97 (sh. 564 m a=5.23 242 mu, a=42.91

.01 N base (alcoholic NaOH)- 292 m a=7.74 580 m a=14.32 610 mp..,a=13.58 (sh.);

and as shown in FIGURE II of the drawing; and

10 (f) has a characteristic infrared absorption spectrum as follows:

3520 1092 (s 2905 (S) (oil) 1065 (S) 2 0 (s) 1038 s 2720 (M) 1010 (S)2660 (M) 980 (S) 1737 (S) 960 (S) 1602 s 940 s 1578 (s) 900 (M) 1560 (M)893 (M) 1456 (S) (oil) 883 (M) 1402 (S) 870 (M) 1377 (S) (oil) 835 (M)1367 (S) 818 (M) 1299 (S) 797 1251 (S) 771 (M) 1241 (S) 7 1224 (S) 723(M) 1197 (S) 712 (M) 1170 (S) 703 (M) 1115 (S) 663 (W) and as shown inFIGURE I of the accompanying drawing.

2. A compound selected from the group consisting of cirolerosusaccording to claim 1, and the acid addition salts thereof.

3. The hydrochloride of the compound defined in claim 1.

4. The sulfate of the compound defined in claim 1.

5. The pictrate of the compound defined in claim 1.

6. The 4'-hydroxyazobenzene-4-sulfonate of the compound defined in claim1.

7. A process which comprises cultivating Streptomyces bellus var.cirolerosus in an aqueous nutrient medium under aerobic conditions untilsubstantial antibiotic activity is imparted to said medium by productionof cirolerosus.

8. A process which comprises cultivating Streptomyces bellus var.cirolerosus under aerobic conditions in an aqueous nutrient mediumcontaining a source of assimilable carbohydrate and assimilable nitrogenuntil substantial antibiotic activity is imparted to said medium byproduction of cirolerosus and isolating the cirolerosus so produced.

9. A process according to claim 8 in which the isolation comprisesfiltering the medium and contacting the filtrate with a water-immisciblesolvent for cirolerosus and recovering cirolerosus from the solventextract.

10. A compound as defined in claim 1, cirolerosus, in its essentiallypure form.

References Cited UNITED STATES PATENTS 3,118,812 1/1964 Gaeumann et a1.167-65 ALBERT T. MEYERS, Primary Examiner. DAREN M. STEPHENS, AssistantExaminer,

1. AN ANTIBIOTIC, ASSAYING AT LEAST 2.5 MCG./MG. OF CIROLEROSUS, ACOMPOUND WHICH (A) IS EFFECTIBVE IN INHIBITING THE GROWTH OFGRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA; (B) IS SOLUBLE IN METHANOL,ETHANOL ETHYL ACETAE, HALOGENATED HYDROCARBONS, ACIDS, BENZENE, GLYCOLS;SLIGHTLY SOLUBLE IN ETHER; AND RELATIVELY INSOLUBLE IN PARAFFINHYDROCARBONS; AND IN ITS ESSENTIALLY PURE FORM (C) HAS THE FOLLOWINGELEMENTAL ANALYSIS: C, 60,90, 60.73;H, 7.08, 6.71;O,28.46;N,2.80; (D)HAS A MOLECULAR WEIGHT OF 974+30 (FROM EQ. WT.); (E) HAS ACHARACTERISTIC ULTRAVIOLET ABSORPTION SPECTRUM AS FOLLOWS:
 7. A PROCESSWHICH COMPRISES CULTIVATING STREPTOMYCES BELLUS VAR. CIROLEROSUS IN ANAQUEOUS NUTRIENT MEDIUM UNDER AEROBIC CONDITIONS UNTIL SUBSTANTIALANTIBIOTIC ACTIVITY IS IMPARTED TO SAID MEDIUM BY PRODUCTION OFCIROLEROSUS.